In vitro antifungal activity of biosynthesized selenium nanoparticles using plant extracts and six comparators against clinical Fusarium strains

Background and Purpose: Fusarium species are commonly resistant to many antifungal drugs. The limited therapeutic options available have led to a surge of research efforts aimed at discovering novel antifungal compounds in recent decades. This study aimed to assess the in vitro antifungal activity of plant-based biosynthesized selenium nanoparticles (Se NPs) and six comparators against a set of clinical Fusarium strains. Materials and Methods: In vitro antifungal activity of Se NPs synthesized using plant extracts of Allium paradoxum, Crocus caspius, Pistacia vera L. hull, Vicia faba L. hull and Heracleum persicum, as well as six common antifungal drugs, namely voriconazole, itraconazole, amphotericin B, posaconazole, natamycin, and caspofungin were evaluated against 94 clinical Fusarium strains using broth microdilution according to Clinical and Laboratory Standards Institute guideline. Results: The obtained results were intriguing since all five types of biosynthesized Se NPs demonstrated significantly higher antifungal activity, compared to antifungal drugs. It was found that Se NPs synthesized by V. faba L. hull extract (0.03 μg/ml) had the lowest geometric mean minimum inhibitory concentration value followed by Se NPs synthesized by P. vera L. hull extract (0.25 μg/ml), A. paradoxum extract (0.39 μg/ml), C. caspius extract (0.55 μg/ml), and H. persicum extract (0.9 μg/ml). Conclusion: Plant-based Se NPs demonstrated supreme antifungal activity and could be considered promising antifungal agents for Fusarium infections. However, tests, such as toxicity and in vivo tests are needed before the product can be used in clinical settings.


Introduction
ungal pathogens pose a serious threat to public health, affecting at least 1.5 million people worldwide each year [1,2].Resistance to conventional antifungal drugs has emerged as a global public health concern, severely limiting available treatment options [3][4][5][6].Furthermore, since fungal and human cells are so similar, it is difficult to discover and develop new and effective antifungal drugs [7,8].
Fusarium is a large and remarkably diverse genus of saprophytic fungi found throughout nature [9].Fusarium species are important phytopathogenic and mycotoxin-producing fungi [10,11].They are also opportunistic fungi that cause a wide range of infections, from superficial and localized infections, such as onychomycosis and keratitis in healthy people to fatal systemic infections in severely ill people [12][13][14].
In the United States and Europe, Fusarium is the second most common mold-causing human infection [15][16][17].This fungal genus has high levels of intrinsic resistance to commonly available antifungal agents, posing a serious challenge to healthcare systems all over the world [15,17].Since Fusarium species are frequently resistant to many antifungal drugs, and since there is a limited therapeutic repository for these fungal infections, research focused on finding novel and potent antifungal compounds as well as alternatives are urgently needed [12,[18][19][20].
F Curr Med Mycol, 2023, 9(4): 17-23 Development of nanotechnology-based therapies in recent years has opened up new avenues for the treatment of drug-resistant fungal infections.Due to their nanometer size and large surface areas, nanoparticles have unique physicochemical properties that increase interactions with microbial cells and significantly influence their antimicrobial effects [21,22].
Selenium is a trace element that is essential for human and animal health, and it is involved in antioxidant defense, metabolism, and detoxification.Due to their low toxicity, high biodegradability, and bioavailability as well as anticancer, antidiabetic, antioxidant, antibacterial, antiprotozoal, antiviral, antifungal, and antibiofilm properties, selenium nanoparticles (Se NPs) are among the most appealing nanomaterials in biomedicine [23,24].
According to some studies, the antimicrobial activity of Se NPs is often attributed to the generation of reactive oxygen species, which can damage the DNA and cell membrane.However, the precise antimicrobial mechanisms of Se NPs have not been fully elucidated, and more research is needed.According to previous research, Se NPs can be synthesized through chemical, physical, or biological processes [25][26][27][28].However, green synthesis or biologically synthesized Se NPs using microorganisms or plant extracts as natural reducing agents provides novel, simple, low-cost, nontoxic, and environment-friendly methods for the production of Se NPs [29][30][31][32].
Some plants contain a high concentration of phytochemical compounds with important biological and medicinal properties.The efficiency and capability of these compounds as nanoparticle reductants in green synthesis have previously been demonstrated [31][32][33].The current study aimed to assess the in vitro antifungal activity of Se NPs made from extracts of Allium paradoxum, Crocus caspius, Pistacia vera L. hull, Vicia faba L. hull, and Heracleum persicum, as well as six antifungal drugs, namely, voriconazole, itraconazole, amphotericin B, posaconazole, natamycin, and caspofungin against a collection of clinical Fusarium strains.

Synthesis of plant-based selenium nanoparticles
The plant-based Se NPs used in this study were synthesized by the Department of Medicinal Chemistry at the School of Pharmacy and Pharmaceutical Sciences Research Centre of Mazandaran University of Medical Sciences, Sari, Iran [31,32].The plants A. paradoxum, C. caspius, P. vera L. hull, V. faba L. hull, and H. persicum were dried in daylight, cut into small pieces (2-3 mm), and stored at room temperature.Each plant (10 g) was combined with 80 mL of deionized water and heated at 50 °C for 1 h.The mixture was then sonicated for 30 min and filtered through the Whatman filter paper.In the process of Se NPs biosynthesis using plant extracts, 17.3 mg of Na2SeO3 was dissolved in 10 mL of deionized water and stirred at 45-50 °C and 500 rpm.Subsequently, 5 mL of the aqueous extract was added to the reaction mixture drop by drop.After two days, the color of the reaction changed from colorless to reddish, indicating the reduction of Se ions.
The final concentrations of agents in the wells ranged from 0.016 to 16 μg/ml for V-Se NPs, A-Se NPs, P-Se NPs, C-Se NPs, H-Se NPs, amphotericin B, voriconazole, natamycin, posaconazole, itraconazole, and 0.008 to 8 μg/ml for caspofungin.
The Fusarium isolates were cultured on Sabouraud dextrose agar (Difco Laboratories, Detroit, MI, USA) and incubated at 35 °C for 5 to 7 days for adequate sporulation.Suspensions were diluted at 1:50 in RPMI 1640 medium to obtain final inoculum between 0.4 × 10 4 to 5 × 10 4 CFU/ml.Plates were incubated at 35 °C, and minimum inhibitory concentrations (MICs) and minimum effective concentrations (MECs) were read after 48 h.
The MIC was determined visually as the lowest concentration of agent that resulted in 100% inhibition of fungal growth while for caspofungin MEC was microscopically determined as the lowest concentration of drug that resulted in the growth of compact hyphal forms compared with growth control.Candida krusei (ATCC 6258), Candida parapsilosis (ATCC 22019), and Aspergillus flavus (ATCC 2004304) served as quality control strains.

Results
All Fusarium strains were previously identified using TEF1 partial gene analysis [34].
The obtained results were extremely intriguing since all five types of biosynthesized Se NPs demonstrated significantly higher antifungal activity, compared to routine antifungal drugs in the clinical settings.

Discussion
Fusarium is a globally distributed multidrugresistant genus that has the potential to cause a wide range of infections in humans [19].In the present study, the inhibitory activity of five plant-based biosynthesized Se NPs and six common antifungal drugs were tested against 98 clinical Fusarium strains.Remarkably, it was discovered that the antifungal activity of plant-based Se NPs was superior to that of voriconazole, itraconazole, amphotericin B, posaconazole, natamycin, and caspofungin in vitro.
Selenium has antimicrobial properties and has been shown to inhibit the growth of fungi and bacteria.Selenium derivatives, such as selenium sulphide, are commonly used in the treatment of pityriasis versicolor [36][37][38][39][40]. Nanotechnology advancements in recent years have provided a safe strategy for the reduction of selenium toxicity.When compared to inorganic and organic forms, Se NPs have lower toxicity and higher bioavailability [24,[41][42][43].
Several studies have shown that biogenic selenium nanoparticles have antifungal activity [29,44,45].Shakibaie et al. investigated the antifungal activity of Bacillus species synthesized selenium nanoparticles against A. fumigatus and Candida albicans, and the measured MICs for C. albicans (70 μg/ml) and A. fumigatus (100 μg/ml) showed that the biogenic Se NPs had good antifungal activity [44].Shahverdi et al. also investigated the antifungal activity of Klebsiella pneumoniae-produced selenium nanoparticles against clinical isolates of the Malassezia and Aspergillus genus.The MICs for all fungal strains were within the range of 10-260 g/ml, with M. sympodialis, M. furfur, and A. terreus showing the highest antifungal activity [45].
Another study assessed the antifungal activity of Se NPs synthesized by Lactobacillus acidophilus in controlling wheat crown and root rot diseases caused by Fusarium species, and biogenic Se NPs successfully inhibited fungal growth at concentrations ranging from 20 to 40 μg/ml [46].Furthermore, several studies on the synthesis of nanoparticles using plant extracts have been published [47][48][49].
There have been few studies on the antifungal activity of biosynthesized Se NPs derived from plant extracts.Gunti  Se NPs derived from Emblica officinalis fruit extract had strong antifungal activity.The MIC values were found to be between 07.50 ± 1.32 and 25.50 ± 2.78 μg/ml.The lowest and highest found MIC values were 07.50 ± 1.32 μg/ml against Rhizopus stolonifer and 25.50 ± 2.78 μg/ml against A. oryzae, respectively [50].
In another study, Kokila et al. biosynthesized Se NPs from Diospyros montana extract and reported antimicrobial activity in the form of zone of inhibition values of 08, 07, and 08 mm against Staphylococcus aureus, Escherichia coli, and A. niger, respectively [51].Ali et al. also investigated the antifungal activity of Se NPs extracted from Capparis decidua fruit against C. albicans and discovered that biosynthesized Se NPs have a high antifungal activity [52].
In the present study, all five biosynthesized Se NPs derived from plant extracts outperformed the commonly used antifungal drugs.Biosynthesized Se NPs, particularly nanoparticles made from V. faba L. hull, P. vera L. hull, and A. paradoxum plant extracts demonstrated potent antifungal activity against clinical Fusarium isolates in vitro.In addition to the antifungal properties of metal ions and the size of the nanoparticles, the antifungal activity of Se NPs in vitro may be attributed to the phytochemical composition of the plant extracts used for green synthesis, as these factors may affect their bioavailability and antimicrobial activity [30,32].

Conclusion
Plant-based Se NPs demonstrated supreme antifungal activity and could be considered promising antifungal agents for Fusarium infections.However, tests, such as toxicity and in vivo tests, are needed before the product can be used in clinical settings.

Table 1 .
Sources and the number of the Fusarium isolates based on the species complexes

Table 3 .
Minimum inhibitory concentrations of five Se NPs and six antifungal drugs on 94 clinical Fusarium isolates